Abstract The long term objective of this proposal is to understand molecular basis of integrin-mediated cell adhesion with the focus on talin ? a master regulator of cell adhesion. Well-known as a physical linker between integrin and actin filament, talin has been extensively studied for more than three decades. The past 15 years have seen a significant advance where talin was found to have the capacity to activate integrin - a key step for initiating all integrin-mediated cell adhesive events such as cell spreading and migration. Using NMR spectroscopy and cell biology approaches (in collaboration with project leader 1), we were the first to show that talin utilizes its N-terminal head (talin-H) to break a cytoplasmic clasp of platelet integrin alphaIIbbeta3 to trigger conformational change and activation of the receptor. We further discovered how talin itself is regulated through an autoinhibition mechanism where a key integrin binding site on talin-H is masked in inactive talin. These findings, together with data from other laboratories, have paved the way for understanding a conformation-based regulation of talin in mediating integrin activation. However, the central issue of how talin spatiotemporally engages with integrin and actin to regulate dynamic cell adhesion is still elusive. Specifically: (i) what mechanism(s) activates talin to bind integrin? (ii) How does the activated talin trigger integrin transmembrane signaling? (iii) How does talin cooperate with kindlin ? another key regulator of integrin activation? Finally, (iv) How is talin regulated to dynamically link integrin to actin filament? Despite an extensive literature, the answers to these fundamental questions are still speculative and controversial. With strong preliminary data, we propose to use structural biology as a major tool coupled with functional analyses to obtain definitive answers to these perplexing questions. Longstanding collaboration with other project leaders for functional experiments is the strength of this proposal, which has proven to be highly productive in the past. We believe the newly proposed studies will lead to a new advance for understanding the mechanisms of integrin signaling and cell adhesion. Given the critical involvement of talin and its associated proteins in thrombosis, stroke, cancer and other diseases, our studies may be also valuable for development of new therapeutics to treat these diseases.